1. Field of the Invention
The invention is directed to systems, compositions and methods for the expression and purification of lipoxygenases, to amino acid and nucleic acid sequences of all or portions of lipoxygenases, to molecular constructs for the expression of lipoxygenases, and, in particular, to methods for the large scale production and use of lipoxygenases in food products.
2. Description of the Background
Lipoxygenases (LOXs; EC1.13.11_), also known as lipoxydases, are non-heme iron-containing dioxygenases distributed in plants and animals. LOXs catalyze hydroperoxidation of polyunsaturated fatty acids in the first step of fatty acid metabolite synthesis, to produce an unsaturated fatty acid hydroperoxide. A LOX definition according to enzyme classification is linoleate: oxygen oxidoreductase (for plant LOX) and arachidonate: oxygen oxidoreductase (for mammalian LOX). In plants, the most common LOX substrates linoleic acid and linolenic acids are converted into a variety of bioactive mediators involved in plant defense, senescence, seed germination, as well as plant growth and development (Grechkin A. Recent developments in biochemistry of the plant lipoxygenase pathway; Prog Lipid Res. 1998 November 37(5):317-52). Lipoxygenases with different specificities, subcellular location, and tissue-specific expression patterns have been identified as ubiquitously found across kingdoms from bacteria to mammals.
LOXs are of commercial value in various industries including but not limited to food-related applications in food processing including bread making (bleaching and improved texture), aroma and flavor enhancement as well as for production of perfumes, paint driers (lipoxygenases: potential starting biocatalysts for the synthesis of signaling compounds. Joo YC, Oh DK. 2012) and pitch control in softwood pulp (Microbial and enzymatic control of pitch in the pulp and paper industry, Ana Gutiérrez & José C. del Río & Angel T. Martínez, Appl Microbiol Biotechnol (2009) 82:1005-4018). Lipoxygenase is present in seeds (e.g. soybeans), grains and many other plant tissues. In the presence of oxygen, lipoxygenase oxidizes unsaturated fatty acids and produces lipid hydroperoxides, which improve dough structure through the oxidation of unsaturated fatty acids and subsequently react with specific chemical components of flour. As a consequence, dough stability and rising is increased, which either or together can increase the volume of the final product.
Regarding the processing of bread, lipoxygenase enzymes offer an advantage over current chemical additives. The flour ingredient industry had long been using chemical bleach, mostly benzoyl peroxide (BPO). Because of potential health concerns over BPO, some Euro countries and China banned the usage of BPO in flour. In the U.S., BPO is still widely used, but the demand keeps shirking although there is currently no safe alternative. Azodiformamide is another chemical alternative, but the dosage is limited to 40 ppm. At this trace dosage, the bleaching effect is quite restrained. In contrast, enzyme additives especially LOXs can replace chemicals to allow for the processing of flour, resulting in the bleaching of bread and its improved texture. In addition, lipid hydroperoxidases decolorize dough and oxidizes carotinoids, converting them into colorless compounds. This blanching of the dough results in lighter colored product, which is highly desired.
With regard to enzymes employed in the food industry, regulations frequently require enzymes to be recognized or proven as safe for use. In the case of lipoxygenases, considering that they are ubiquitously found in plants and consumed by humans and animals alike, plant lipoxygenases are considered safe for use, and therefore, of major value to the industry. Although soy extracts containing high levels of lipoxygenases have been used as an additive for bread manufacturing, soy produces an undesirable taste and smell and, accordingly, not often a useful option.
Because of plant LOX value, many attempts at high-level expression of recombinant plant derived LOX from soy, rice, potato and other sources by heterologous expression in microbial hosts including, but not limited to bacteria such as E. coli (BL21 strain), Bacilli, and in yeast has been attempted, though production was limited [3-8]. The best of these, although still a poor expression from E. coli, was observed at very cold temperatures [8]. Only one lipoxygenase was produced in Bacilli at high-levels (˜160 mg/L), but the lipoxygenase was from a bacterial enzyme, not a plant and consequently not approved for use in the human food industry [9, 10]. In addition, yields still could not achieve desired levels. Accordingly, a need exists for high level expression of plant lipoxygenases that is generally recognized as safe for use in foods, and easily produced in large quantities.